X-ray-activated polymerization expanding the frontiers of deep-tissue hydrogel formation

Photo-crosslinking polymerization stands as a fundamental pillar in the domains of chemistry, biology, and medicine. Yet, prevailing strategies heavily rely on ultraviolet/visible (UV/Vis) light to elicit in situ crosslinking. The inherent perils associated with UV radiation, namely the potential for DNA damage, coupled with the limited depth of tissue penetration exhibited by UV/Vis light, severely restrict the scope of photo-crosslinking within living organisms. Although near-infrared light has been explored as an external excitation source, enabling partial mitigation of these constraints, its penetration depth remains insufficient, particularly within bone tissues. In this study, we introduce an approach employing X-ray activation for deep-tissue hydrogel formation, surpassing all previous boundaries. Our approach harnesses a low-dose X-ray-activated persistent luminescent phosphor, triggering on demand in situ photo-crosslinking reactions and enabling the formation of hydrogels in male rats. A breakthrough of our method lies in its capability to penetrate deep even within thick bovine bone, demonstrating unmatched potential for bone penetration. By extending the reach of hydrogel formation within such formidable depths, our study represents an advancement in the field. This application of X-ray-activated polymerization enables precise and safe deep-tissue photo-crosslinking hydrogel formation, with profound implications for a multitude of disciplines.


Supplementary Note 2: Instruments
The X-ray-excited fluorescence emission spectra were recorded using an Andor SR-500i spectrometer (Andor Technology Co. Belfast, UK) equipped with a Hamamatsu R928 photomultiplier.
TGA was performed on a TA Q50 thermal gravimetric analyzer (TA Instruments, US) under a nitrogen flow.Accurately weighted amounts of samples were heated at a scanning rate of 10 °C min -1 from room temperature to 800 °C.
X-ray photoelectron spectroscopy (XPS) was carried out on a Thermo Scientific ESCALab 250Xi (Thermo Fisher Scientific, US) using 200 W monochromated Al K Alpha radiation.The 500 μm X-ray spot was used for XPS analysis.The base pressure in the analysis chamber was about 3 × 10 Powder X-ray diffraction (PXRD) patterns were recorded on D8 ADVANCE X-ray powder diffractometer system (Bruker Corporation, German).Cu Kα source is used to generate X-ray beam with a wavelength of 1.54 Å, tube voltage of 40 kV, and current of 40 mA.
FTIR spectra were recorded in the region of 4000-400 cm -1 for each sample on a Nicolet iS10 Fourier transform infrared spectrophotometer (Thermo Fisher Scientific, US).Samples were previously grounded and mixed thoroughly with KBr.The spectrum for each sample was obtained from averaging 32 scans over the selected wavenumber range.
The morphological characterizations, including transmission electron microscope (TEM) observations, energy dispersive X-ray spectroscopy (EDX) maps and high-angle annular dark-field (HAADF) Scanning TEM (STEM) images were performed by using a F200x FEI TalosF200x scanning transmission electron microscope (Thermo Fisher Scientific, US). 13 C solid-state nuclear magnetic resonance (NMR) spectra were obtained on a Bruker 400 M spectrometer (Bruker Corporation, German). 13C NMR and 1 H NMR spectra were performed on a Bruker AV600 spectrometer (Bruker Corporation, German) or a QOne WNMR-I-400MHz spectrometer (Zhongke-Niujin, China) using tetramethylsilane as internal standard.
Gel permeation chromatography (GPC) (Shimazu, Japan) equipped a refractive index detector was used to determine the elution curves of the samples in parallel experiments.The column was placed under 30°C; the system was operated at a flow rate of 1.0 mL min -1 with tetrahydrofuran as an eluent.Polystyrene (Mp = 2,755 g mol -1 ) was used as standards for the calibration.
AR2000ex-type rotational rheometer (TA, US) was used to investigate the rheological property.

Supplementary Discussion 1: Analysis for monomer conversions
NMR analysis was performed further to evaluate the monomer conversion at different irradiation intervals.A mono-vinyl olefin PEGMA is used to replace of PEGDA.Briefly, PEGMA, triethanolamine, complex of camphorquinone and methyl-β-cyclodextrin (MCD/CQ), and HNTs-based X-ray-activated visible persistent luminescent emitting phosphors (HNTs@YF3:Tb 3+ ) were added into D2O and then exposed to X-ray at different times.Other conditions are the same as the methods part in main text except for changing the irradiation times.After easing of irradiation, the samples were placed at room temperature and kept in a dark place for 24 h.Then the samples were subjected to NMR directly.
A comparison among the 1 H NMR spectra of PEGMA, triethanolamine, and MCD/CQ is made to pick out the peaks for the conversion calculation.As shown in Supplementary Fig. 15, the signals from the protons in vinyl groups (Ha1, Ha2, and Hb) and the proton in methylene group (Hc) that closed to ester bond do not overlap with those from triethanolamine or MCD/CQ.The vinyl groups can be consumed after the free radical polymerization of PEGMA, which would give rise to a decrease in the integral values from Ha1, Ha2, and Hb.Meanwhile, the integral value of Hc cannot changed because the methylene groups did not participate in the free radical polymerization.Therefore, the integral values from Ha1, Ha2, Hb, and Hc are used to calculate the monomer conversion (%): where Ia1, Ia2, Ib, and Ic represent the integral values from Ha1, Ha2, Hb, and Hc respectively.The results show that the monomer conversion (%) calculated as 5.3%, 45.0%, 46.0%, and 49.0% as the irradiation time ranging from 1 min, 3 min, 5 min, and 10 min, respectively (Supplementary Fig. 17).The monomer conversion (%) increases with the increase of the irradiation time.A significant difference can be found between the monomer conversion (%) of 1 min and 3 min.After 3 min, the monomer conversion (%) changes slowly with the continuous increase of irradiation time.
Additional parallel experiments were also conducted by using PEGMA to investigate whether the polymerization behavior can take place by using these components: v: PEGMA; vi: PEGMA, MCD/CQ, and triethanolamine; vii: PEGMA and HNTs@YF3:Tb 3+ ; viii: PEGMA, MCD/CQ, and HNTs@YF3:Tb 3+ .The monomer conversions (%) for the above-mentioned groups (v, vi, vii, and viii) are all calculated as ca.0% based on the NMR results shown in Supplementary Fig. 16, indicating the polymerization cannot take place when using the above-mentioned groups containing different components.The results match well with the findings from GPC curves.The radical cannot be generated from X-ray/CQ or bypassing the composite, and the co-initiator triethanolamine is also essential for the Xcrosslinking system.Only combination of all required components, along with X-ray activation of HNTs@YF3:Tb 3+ , can lead to successful free radical polymerization.

Supplementary Discussion 2: Influence of molecular weight on the gelation behaviors
The impact of PEGDA's molecular weight (200, 400, and 600 g mol -1 ) and content on gelation time and rheological properties were investigated.Here, we use PEGDA200, PEGDA400, and PEGDA600 to represent PEGDA with molecular weights of 200, 400, and 600 g mol -1 , respectively.The contents are the same as the reaction conditions used in in vitro gelatinization study except for changing the irradiation times.PEGDA200, PEGDA400, and PEGDA600 can be gelated within 60 s, 180 s, and 360 s, respectively, suggesting the gelation time increases with the increase of the PEGDA's molecular weight.However, it should be noted that PEGDA200 cannot be dissolved in water, which yields a nonuniform hydrogel (Supplementary Fig. 13a) and is not considered for further studies.PEGDA400 and PEGDA600 can be dissolved in water at any proportion.The obtained PEGDA400 and PEGDA600 hydrogel samples (Supplementary Fig. 13b&c) were cut into desired sizes and then subjected to rheological analysis.The rheological characteristics of the resulting hydrogel were demonstrated by the storage modulus (G') being greater than the loss modulus (G'') over an average frequency range of 1 to 100 rad s -1 (Supplementary Fig. 13d).The PEGDA600 hydrogel shows higher G' and G'' than the PEGDA400 hydrogel.
The main purpose of this study is to develop an X-crosslinking approach for potential in vivo use.So, we regarded PEGDA400 as the optimal component because it can be dissolved in water and is able to achieve a gelation state in a shorter time than that of a higher molecular weight.Furthermore, the PEGDA400 content (20, 30, and 40%) on gelation time was examined.HNTs@YF3:Tb 3+ cannot achieve good dispersity when the PEGDA400 content is higher than 50%.The results indicate that the gelation can be achieved within 600 s, 360 s, and 180 s as the PEGDA400 content ranges from 20, 30, to 40%, respectively, implying the gelation time decreases as the increase of PEGDA400 content and the content of 40% was used for further investigations.

Supplementary Discussion 3: Study of swelling behaviors
The swelling behavior of the synthesized hydrogels was measured by referring to the method reported by the literature. 1 The obtained hydrogel was dialyzed against deionized water thoroughly to remove the unreacted residues and then dried at room temperature.An accurately weighed dry hydrogel piece (W0) was placed into deionized water at room temperature.The swollen samples were picked out from the medium at specific time intervals, wiped, weighed (Wi), and then placed back into the medium.The measurements were carried out in three parallel groups to calculate the mean value and standard deviation.
The swelling degree (SDt) at various time intervals was calculated as: where i represents the swollen time, Wi represents the weight of the swollen sample at different times and W0 is the original weight of the dry hydrogel piece.The swelling behaviors were monitored until the Wi reached a constant value which is defined as the equilibrium swelling degree (ESD).
The Fickian diffusion model was employed to gain insight into the swelling kinetics.where t and k represent time and a constant, respectively; Mt and M∞ are the amount of solvent absorbed at time t (h) and at equilibrium, respectively; n is the swelling exponent, also known as the swelling exponent, is calculated based on the slope of log (SDt/ESD) vs logt (0 < SDt/ESD ≤ 0.6).
The swelling behavior of the hydrogels was carefully investigated by plotting SDt vs time (Supplementary Fig. 14a).SDt increases rapidly at the first 50 min and then reaches a plateau after 120 min.The ESD is calculated as 330%.The swelling exponent (n) is calculated as 0.400, suggesting a Fickian diffusion mechanism. 2

Supplementary Discussion 4: Influence of X-ray irradiation time on swelling behaviors
Generally, the degree of hydrogel crosslinking holds a negative correlation with swelling behaviors. 3For the hydrogels made from the same monomers, a higher equilibrium swelling degree (ESD) means a lower degree of hydrogel crosslinking.So, we investigated the effect of radiation time (200 s, 400 s, and 600 s) on swelling behavior in order to reveal the effect on the degree of hydrogel crosslinking.The contents and X-ray source are the same as those used in in vitro gelatinization study except for changing the irradiation times.
SDt reaches the plateau after 120 min, 300 min, and 500 min as for the case of 600 s, 400 s, and 200 s, respectively (Supplementary Fig. 14a).The ESD values are calculated as 435 %, 352 %, and 330 % as the radiation time increases from 200 s to 600s (Supplementary Fig. 14b).The results suggest that a longer radiation time can give rise to a lower ESD value and a slower swelling behavior of the obtained hydrogel, implying the degree of hydrogel crosslinking can be well-controlled by modulating the radiation time.

Supplementary Discussion 5: Influence of the tissue depth on the radioluminescence
We have investigated the influence of the tissue depth on the radioluminescence behaviors of the prepared HNTs@YF3:Tb 3+ .After exposure to X-ray (40 kV, 30 mA) for 10 min, the peak intensity at 545 nm can reach a constant value and was recorded as I0.Then the tissue samples with a certain thickness were placed between the X-ray source and HNTs@YF3:Tb 3+ sample.The peak intensity at 545 nm was recorded as It after it reached the plateau.The intensity ratio (It / I0) was calculated and the relationship between intensity ratio and tissue thickness is shown in Supplementary Fig. 21.Two different kinds of tissues from chicken breast and bovine bones were used in this study.
The results indicated that the intensity ratio decreased with the increase in tissue thickness, in which the intensity ratio decreased more significantly across bone tissue than across soft tissue.We have demonstrated the gelatinization capability of our approach to enable the formation of photo-crosslinking hydrogels within thick bone tissue of ca.7 mm.Such a high penetrability can satisfy most cases for in vivo uses.The penetrability of the proposed Xcrosslinking system is significantly higher than those of other cases including UV, visible light, and near-infrared light.A higher energy X-ray may be helpful to achieve higher penetrability.

Supplementary Discussion 6: Study of swelling behaviors of the solidified hydrogel samples
Additionally, the swelling behaviors of the original solidified hydrogel samples without the dialysis and drying procedures are also investigated.To make the experiment design align with the actual application conditions, the swelling behaviors of the hydrogel samples prepared following the on-off-on circulation process were investigated in phosphate buffer saline (PBS) of pH=7.4 at 37 °C.The accurately weighed original hydrogel sample (m0) was directly placed into the medium.The swollen samples were picked out from the medium at specific time intervals, wiped, weighed (mi), and then placed back into the medium.The measurements were carried out in three parallel groups to calculate the mean value and standard deviation.
The swelling degree (SDt*) of the original solidified hydrogel samples at various time intervals was calculated as: where i represents the swollen time, mi represents the weight of the swollen sample at different times, and m0 is the original weight of the hydrogel sample.The swelling behaviors were monitored until the mi reached a constant value which is defined as the equilibrium swelling degree measured from the solidified hydrogel sample (ESD*).
The swelling behaviors of hydrogel samples prepared following the on-offon circulation process are depicted in Supplementary Fig. 22.The sample prepared after 10 cycles (1.5 mGy) shows an ESD* of ca.5][6] Moreover, the ESD* can be further decreased by extending the cycle times, which is beneficial to meet different requirements of bio-medical applications

Supplementary Discussion 7: Potential application scenarios of the proposed Xcrosslinking strategy
Visible light-cured dental resin has been commonly employed for repairing teeth defects in past decades, which affords a safer way for human bodies than ultraviolet-curing resin. 7Though the visible light-cured dental resin can satisfy the majority of requirements for repairing teeth defects in clinical, the conventional visible light-cured strategy still suffers from the limited penetration issues in deep defects.The light intensity is distributed in a gradient decrease in the thickness direction, resulting in heterogeneity of conversion in the cured material. 8,9As a result, the degree of conversion of the photopolymerization decreases as the increased filter thickness of dental resin filled in the defects. 10,11For most of commercial visible light-cured dental resin, the penetration depth is usually limited to 1~3 mm. 12,13Once the visible light-cured dental resin used to repair deep defects, the inhomogeneous irradiance may result in inadequate polymerization, shrinkage, and corresponding shrinkage stress as well as elution of low molecular substances. 14Therefore, sometimes, visible light-cured dental resin is not recommended to be directly used in the teeth with defects exceeding 2 mm. 15,16More operations and additional materials may be required for repairing the deep defects, which means added discomfort and a delay in patient's mouth opening time.The proposed Xcrosslinking strategy may be beneficial to break the limitation of the visible light-cured dental resin in deep defects.X-ray-activated visible persistent luminescent emitting phosphors (X-PLNPs) can serve as micro-lamps dispersed in the resin matrix rather than conventional external planar light sources, the degree of conversion in the product afforded by the former case should not be easily affected by the thickness, and enhanced mechanical properties are also expected. 17The Xcrosslinking strategy may be a promising supplementary technique to visible light-cured dental resin for repairing deep defects..20 a HNTs is abbreviated from halloysite nanotubes; HNTs@YF3:Tb 3+ is abbreviated from halloysite nanotubes-based X-ray-activated visible persistent luminescent emitting phosphors.b overlapped with the peak of (121) in YF3 c overlapped with the peak of (321) in YF3 PEGMA.Peaks labelled as b (blue) and c (blue) represent the hydrogen b (blue) and c (blue) in the structure of PEGMA, respectively.b 1 H NMR spectrum of the sample from group vi derived from the solution containing PEGMA, complex of camphorquinone and methyl-β-cyclodextrin (MCD/CQ), and triethanolamine.c 1 H NMR spectrum of the sample from group vii derived from the solution containing PEGMA and halloysite nanotubes-based X-rayactivated visible persistent luminescent emitting phosphors (HNTs@YF3:Tb 3+ ).d 1 H NMR spectrum of the sample from group viii derived from the solution containing PEGMA, MCD/CQ, and HNTs@YF3:Tb 3+ .halloysite nanotubes-based X-ray-activated visible persistent luminescent emitting phosphors (HNTs@YF3:Tb 3+ ), poly(ethylene glycol) methyl ether acrylate (PEGMA), triethanolamine, and the complex of camphorquinone and methyl-β-cyclodextrin (MCD/CQ) with exposure to X-ray for different times.a 0 min.Peaks labelled as a1 (blue) and a2 (blue) represent the hydrogen a in the structure of PEGMA.Peaks labelled as b (blue) and c (blue) represent the hydrogen b (blue) and c (blue) in the structure of PEGMA, respectively.b 1 min.c 3 min.d 5 min.e 10min.

Supplementary
log  + log  Supplementary Equation(4) -10mbar.Typically the hydrocarbon C 1s line at 284.8 eV from adventitious carbon was used for energy referencing.Other acquisition parameters: Number of Scans: 5; Lens Mode: Standard; Analyser Mode: CAE: Pass Energy 30.0 eV; Energy Step Size: 0.050 eV; Number of Energy Steps: ca.400.

Table 1 .
X-ray photoelectron spectroscopy data a HNTs@YF3:Tb 3+ is abbreviated from halloysite nanotubes-based X-ray-activated visible persistent luminescent emitting phosphors.b JCPDS is abbreviated from Joint Committee on Powder Diffraction Standards. a

Table 3 .
The parameters of the peaks in the X-ray diffraction patterns a